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Site specific silencing of caveolin acylation causes accumulation of Alexa labeled Cholera Toxin B chain at distinct sites in the lipid raft‐caveolae endosomal system in polarized HepG2 cells
Author(s) -
Everson William V.,
Carter Josh,
Kentucky Eric J Smart.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1180.4
Subject(s) - caveolae , endocytosis , endocytic cycle , microbiology and biotechnology , endosome , lipid raft , ganglioside , alexa fluor , cholera toxin , chemistry , biology , biochemistry , intracellular , cell , signal transduction , fluorescence , physics , quantum mechanics
We have previously shown that silencing of the three distinct acylation sites in caveolin‐1 by substitution of alanine for cysteine at positions 133 (cav 133), 143 (cav 143) and 156 (cav 156) alter the cholesterol transport activity of 2 distinct cytosolic protein complexes. In this study, we examined the effect of these acylation mutants on endocytosis of fluorescent tagged cholera toxin B chain (CT‐B), which binds to the ganglioside GM1 and marks lipid raft/caveolae endocytosis. We stably expressed wild type caveolin (wt cav) or each of the single site acylation mutants in HepG2 cells, a cell line which lacks detectable expression of caveolin. Cells were cultured with Alexa 488 labeled wheat germ agglutinin (WGA), a marker of the constitutive endocytic pathway, and Alexa 555 labeled CT‐B. Ligand uptake was followed by live confocal microscopy at 37 deg or cells were incubated for time points up to 4 hours at 4 deg or 37 deg, fixed and immunostained for caveolin, organelle markers, or annexins (proteins associated with lipid raft/caveolae trafficking). Expression of each of the acylation mutants showed accumulation of CT‐B in distinct intracellular sites compared to cells expressing wt cav. Expression of the mutants also altered the distribution and accumulation of WGA indicating that alteration in caveolae/lipid raft trafficking impacts the constitutive endocytic system. Supported by NIH.